Immune checkpoint inhibition (CPI) with anti-PD-1/PD-L1 antibodies has changed the treatment landscape for several types of cancers including bladder cancer. In patients with advanced bladder cancer, CPI can induce unprecedented durable responses. However, only a subset of patients responds to such treatment necessitating a better understanding of mechanisms of primary resistance to facilitate the identification of predictive biomarkers and rational combination approaches. We used an integrative approach to identify biomarkers in a large clinical trial cohort of patients with metastatic bladder cancer that are independently associated with response/resistance to CPI beyond tumor mutational burden (TMB) alone. We identified three key gene modules derived from pre-treatment tumor transcriptomic data: a ?good immune? module associated with response to CPI, a stromal module associated with resistance to CPI, and a ?bad immune? module also associated with resistance to CPI, but that appears to mediate the negative impact of the stromal module upon outcomes with CPI. Using single cell RNA sequencing data generated from fresh bladder cancer specimens to dissect the cellular components and molecular interactions responsible for expression of these 3 gene modules, we determined that the ?good immune? module emanates largely from T- cells and NK cells, the stromal module emanates from cancer-associated fibroblasts (CAFs), and the ?bad immune? module emanates predominantly from monocytes-macrophages (M?). We hypothesize that myeloid cells drive primary resistance to CPI in bladder cancer. We further hypothesize that comprehensive cellular and molecular maps of bladder cancer will facilitate identification of precise monocyte-M? subpopulations, and CAF-myeloid-T-cell interactions, that can be leveraged to define novel biomarkers and therapeutic targets. With access to a unique set of clinical trial cohorts that will provide tumor tissue, blood and urine specimens as well as outcome data to CPI, we are uniquely positioned to: (Aim 1) Dissect gene modules associated with response/resistance to CPI using high resolution maps of the cellular and molecular landscape of muscle-invasive bladder cancer; Aim 2: Define the role of ?bad immune? module genes in governing monocyte-M?-mediated immune suppression; Aim 3: Refine and validate a monocyte-M?-related gene signature as a biomarker of CPI resistance in clinical trial cohorts. This proposal comprises an integrated network of multi-disciplinary collaborative investigators (GU oncologists, urologists, immunologists and bioinformaticians) to accelerate translational research and maximize future clinical benefits. These efforts will lead to the discovery of biomarkers, and potential therapeutic targets, to extend the benefits of CPI beyond the 15-20% of patients with advanced bladder cancer that respond to treatment.